Protein corona alleviates adverse biological effects of nanoplastics in breast cancer cells.

Pollution from micro- and nanoplastics (MNPs) has long been a topic of concern due to its potential impact on human health. MNPs can circulate through human blood and, thus far, have been found in the lungs, spleen, stomach, liver, kidneys and even in the brain, placenta, and breast milk. While data are already available on the adverse biological effects of pristine MNPs (e.g. oxidative stress, inflammation, cytotoxicity, and even cancer induction), no report thus far clarified whether the same effects are modulated by the formation of a protein corona around MNPs. To this end, here we use pristine and human-plasma pre-coated polystyrene (PS) nanoparticles (NPs) and investigate them in cultured breast cancer cells both in terms of internalization and cell biochemical response to the exposure. It is found that pristine NPs tend to stick to the cell membrane and inhibit HER-2-driven signaling pathways, including phosphatidylinositol-3-kinase (PI3K)/protein kinase B (AKT) and mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) pathways, which are associated with cancer cell survival and growth. By contrast, the formation of a protein corona around the same NPs can promote their uptake by endocytic vesicles and final sequestration within lysosomes. Of note is that such intracellular fate of PS-NPs is associated with mitigation of the biochemical alterations of the phosphorylated AKT (pAKT)/AKT and phosphorylated ERK (pERK)/ERK levels. These findings provide the distribution of NPs in human breast cancer cells, may broaden our understanding of the interactions between NPs and breast cancer cells and underscore the crucial role of the protein corona in modulating the impact of MNPs on human health.

Enhancing Immune Responses against SARS-CoV-2 Variants in Aged Mice with INDUK: A Chimeric DNA Vaccine Encoding the Spike S1-TM Subunits.

Currently available vaccines against COVID-19 showed high efficacy against the original strain of SARS-CoV-2 but progressively lower efficacy against new variants. In response to emerging SARS-CoV-2 strains, we propose chimeric DNA vaccines encoding the spike antigen, including a combination of selected key mutations from different variants of concern. We developed two DNA vaccines, pVAX-S1-TM-D614G and pVAX-S1-TM-INDUK (INDUK), encoding the SARS-CoV-2 S1 spike subunit in fusion with the transmembrane region that allows protein trimerization as predicted by in silico analysis. pVAX-S1-TM-D614G included the dominant D614G substitution, while the chimeric vaccine INDUK contained additional selected mutations from the Delta (E484Q and L452R) and Alpha (N501Y and A570D) variants. Considering that aging is a risk factor for severe disease and that suboptimal vaccine responses were observed in older individuals, the immunogenicity of pVAX-S1-TM-D614G and INDUK was tested in both young and aged C57BL/6 mice. Two vaccine doses were able to trigger significant anti-SARS-CoV-2 antibody production, showing neutralizing activity. ELISA tests confirmed that antibodies induced by pVAX-S1-TM-D614G and INDUK were able to recognize both Wuhan Spike and Delta variant Spike as trimers, while neutralizing antibodies were detected by an ACE2:SARS-CoV-2 Spike S1 inhibitor screening assay, designed to assess the capacity of antibodies to block the interaction between the viral spike S1 protein and the ACE2 receptor. Although antibody titer declined within six months, a third booster dose significantly increased the magnitude of humoral response, even in aged individuals, suggesting that immune recall can improve antibody response durability. The analysis of cellular responses demonstrated that vaccination with INDUK elicited an increase in the percentage of SARS-CoV-2-specific IFN-γ producing T lymphocytes in immunized young mice and TNF-α-producing T lymphocytes in both young and aged mice. These findings not only hold immediate promise for addressing evolving challenges in SARS-CoV-2 vaccination but also open avenues to refine strategies and elevate the effectiveness of next-generation vaccines.

SIRT6 promotes metastasis and relapse in HER2-positive breast cancer.

The histone deacetylase sirtuin 6 (SIRT6) has been endowed with anti-cancer capabilities in many tumor types. Here, we investigate the impact of SIRT6-overexpression (SIRT6-OE) in Delta16HER2 mice, which are a bona fide model of HER2-positive breast cancer. After an initial delay in the tumor onset, SIRT6-OE induces a more aggressive phenotype of Delta16HER2 tumors promoting the formation of higher number of tumor foci and metastases than controls. This phenotype of SIRT6-OE tumors is associated with cancer stem cell (CSC)-like features and tumor dormancy, and low senescence and oxidative DNA damage. Accordingly, a sub-set of HER2-positive breast cancer patients with concurrent SIRT6-OE has a significant poorer relapse-free survival (RFS) probability than patients with low expression of SIRT6. ChIP-seq, RNA-seq and RT-PCR experiments indicate that SIRT6-OE represses the expression of the T-box transcription factor 3 (Tbx3) by deacetylation of H3K9ac. Accordingly, loss-of-function mutations of TBX3 or low TBX3 expression levels are predictive of poor prognosis in HER2-positive breast cancer patients. Our work indicates that high levels of SIRT6 are indicative of poor prognosis and high risk of metastasis in HER2-positive breast cancer and suggests further investigation of TBX3 as a downstream target of SIRT6 and co-marker of poor-prognosis. Our results point to a breast cancer subtype-specific effect of SIRT6 and warrant future studies dissecting the mechanisms of SIRT6 regulation in different breast cancer subtypes.

The Transformative Potential of Lipid Nanoparticle-Protein Corona for Next-Generation Vaccines and Therapeutics.

The integration of the lipid nanoparticle (LNP)-protein corona as a pioneering approach for the development of vaccines against the present and future SARS-CoV-2 variants of concern marks a significant shift in the field. This concept holds great promise, offering a universal platform that can be adaptable to combat future pandemics caused by unknown viruses. Understanding the complex interactions among the protein corona, LNPs, and receptors is crucial for harnessing its potential. This knowledge will allow optimal vaccine formulations and improve their effectiveness. Safety assessments are essential to ensure suitability for human use, compliance with regulatory standards, and rigorous quality control in manufacturing. This transformative workflow requires collaborative efforts, expanding our foundational knowledge and translating advancements from the laboratory to clinical reality. The LNP-protein corona approach represents a paradigmatic shift with far-reaching implications. Its principles and insights can be leveraged beyond specific applications against SARS-CoV-2, enabling a universal platform for addressing viral threats, cancer, and genetic diseases.

Insights into the effect of polyethylene terephthalate (PET) microplastics on HER2 signaling pathways.

Plastic pollution poses a significant threat to both ecosystems and human health, as fragments of microscale size are daily inhaled and ingested. Such tiny specks are defined as microplastics (MPs), and although their presence as environmental contaminants is ubiquitous in the world, their possible effects at biological and physiological levels are still not clear. To explore the potential impacts of MP exposure, we produced and characterized polyethylene terephthalate (PET) micro-fragments, then administered them to living cells. PET is widely employed in the production of plastic bottles, and thus represents a potential source of environmental MPs. However, its potential effects on public health are hardly investigated, as the current bio-medical research on MPs mainly utilizes different models, such as polystyrene particles. This study employed cell viability assays and Western blot analysis to demonstrate cell-dependent and dose-dependent cytotoxic effects of PET MPs, as well as a significant impact on HER-2-driven signaling pathways. Our findings provide insight into the biological effects of MP exposure, particularly for a widely used but poorly investigated material such as PET.

Mechanistic Insights into the Superior DNA Delivery Efficiency of Multicomponent Lipid Nanoparticles: An In Vitro and In Vivo Study.

Lipid nanoparticles (LNPs) are currently having an increasing impact on nanomedicines as delivery agents, among others, of RNA molecules (e.g., short interfering RNA for the treatment of hereditary diseases or messenger RNA for the development of COVID-19 vaccines). Despite this, the delivery of plasmid DNA (pDNA) by LNPs in preclinical studies is still unsatisfactory, mainly due to the lack of systematic structural and functional studies on DNA-loaded LNPs. To tackle this issue, we developed, characterized, and tested a library of 16 multicomponent DNA-loaded LNPs which were prepared by microfluidics and differed in lipid composition, surface functionalization, and manufacturing factors. 8 out of 16 formulations exhibited proper size and zeta potential and passed to the validation step, that is, the simultaneous quantification of transfection efficiency and cell viability in human embryonic kidney cells (HEK-293). The most efficient formulation (LNP15) was then successfully validated both in vitro, in an immortalized adult keratinocyte cell line (HaCaT) and in an epidermoid cervical cancer cell line (CaSki), and in vivo as a nanocarrier to deliver a cancer vaccine against the benchmark target tyrosine-kinase receptor HER2 in C57BL/6 mice. Finally, by a combination of confocal microscopy, transmission electron microscopy and synchrotron small-angle X-ray scattering, we were able to show that the superior efficiency of LNP15 can be linked to its disordered nanostructure consisting of small-size unoriented layers of pDNA sandwiched between closely apposed lipid membranes that undergo massive destabilization upon interaction with cellular lipids. Our results provide new insights into the structure-activity relationship of pDNA-loaded LNPs and pave the way to the clinical translation of this gene delivery technology.

The protein corona reduces the anticancer effect of graphene oxide in HER-2-positive cancer cells.

In the last decade, graphene oxide (GO)-based nanomaterials have attracted much attention for their potential anti-cancer properties against various cancer cell types. However, while in vitro studies are promising, following in vivo investigations fail to show any relevant efficacy. Recent research has clarified that the wide gap between benchtop discoveries and clinical practice is due to our limited knowledge about the physical-chemical transformation of nanomaterials in vivo. In physiological environments, nanomaterials are quickly coated by a complex dress of biological molecules referred to as the protein corona. Mediating the interaction between the pristine material and the biological system the protein corona controls the mechanisms of action of nanomaterials up to the sub-cellular level. Here we investigate the anticancer ability of GO in SK-BR-3 human breast cancer cells over-expressing the human epidermal growth factor receptor 2 (HER-2), which is functionally implicated in the cell growth and proliferation through the activation of downstream pathways, including the PI3K/AKT/mTOR and MAPK/ERK signaling cascades. Western blot analysis demonstrated that GO treatment resulted in a marked decrease in total HER-2, associated with a down-regulation of the expression and activation of protein kinase B (AKT) and extracellular signal-regulated kinase (ERK) thus indicating that GO may act as a potent HER-2 inhibitor. On the other side, the protein corona reverted the effects of GO on HER-2 expression and molecular downstream events to the control level. Our findings may suggest a mechanistic explanation of the reduced anticancer properties of GO-based nanomaterials in vivo. These results may also represent a good prediction strategy for the anticancer activity of nanomaterials designed for biomedical purposes, reaffirming the necessity of exploring their effectiveness under physiologically relevant conditions before moving on to the next in vivo studies.

HER2-Displaying M13 Bacteriophages induce Therapeutic Immunity against Breast Cancer.

The advent of trastuzumab has significantly improved the prognosis of HER2-positive (HER2+) breast cancer patients; nevertheless, drug resistance limits its clinical benefit. Anti-HER2 active immunotherapy represents an attractive alternative strategy, but effective immunization needs to overcome the patient's immune tolerance against the self-HER2. Phage display technology, taking advantage of phage intrinsic immunogenicity, permits one to generate effective cancer vaccines able to break immune tolerance to self-antigens. In this study, we demonstrate that both preventive and therapeutic vaccination with M13 bacteriophages, displaying the extracellular (EC) and transmembrane (TM) domains of human HER2 or its Δ16HER2 splice variant on their surface (ECTM and Δ16ECTM phages), delayed mammary tumor onset and reduced tumor growth rate and multiplicity in ∆16HER2 transgenic mice, which are tolerant to human ∆16HER2. This antitumor protection correlated with anti-HER2 antibody production. The molecular mechanisms underlying the anticancer effect of vaccine-elicited anti-HER2 antibodies were analyzed in vitro against BT-474 human breast cancer cells, sensitive or resistant to trastuzumab. Immunoglobulins (IgG) purified from immune sera reduced cell viability mainly by impairing ERK phosphorylation and reactivating retinoblastoma protein function in both trastuzumab-sensitive and -resistant BT-474 cells. In conclusion, we demonstrated that phage-based HER2 vaccines impair mammary cancer onset and progression, opening new perspectives for HER2+ breast cancer treatment.

Efficient Delivery of DNA Using Lipid Nanoparticles.

DNA vaccination has been extensively studied as a promising strategy for tumor treatment. Despite the efforts, the therapeutic efficacy of DNA vaccines has been limited by their intrinsic poor cellular internalization. Electroporation, which is based on the application of a controlled electric field to enhance DNA penetration into cells, has been the method of choice to produce acceptable levels of gene transfer in vivo. However, this method may cause cell damage or rupture, non-specific targeting, and even degradation of pDNA. Skin irritation, muscle contractions, pain, alterations in skin structure, and irreversible cell damage have been frequently reported. To overcome these limitations, in this work, we use a microfluidic platform to generate DNA-loaded lipid nanoparticles (LNPs) which are then characterized by a combination of dynamic light scattering (DLS), synchrotron small-angle X-ray scattering (SAXS), and transmission electron microscopy (TEM). Despite the clinical successes obtained by LNPs for mRNA and siRNA delivery, little is known about LNPs encapsulating bulkier DNA molecules, the clinical application of which remains challenging. For in vitro screening, LNPs were administered to human embryonic kidney 293 (HEK-293) and Chinese hamster ovary (CHO) cell lines and ranked for their transfection efficiency (TE) and cytotoxicity. The LNP formulation exhibiting the highest TE and the lowest cytotoxicity was then tested for the delivery of the DNA vaccine pVAX-hECTM targeting the human neoantigen HER2, an oncoprotein overexpressed in several cancer types. Using fluorescence-activated cell sorting (FACS), immunofluorescence assays and fluorescence confocal microscopy (FCS), we proved that pVAX-hECTM-loaded LNPs produce massive expression of the HER2 antigen on the cell membrane of HEK-293 cells. Our results provide new insights into the structure-activity relationship of DNA-loaded LNPs and pave the way for the access of this gene delivery technology to preclinical studies.

Breast Cancer Treatment: The Case of Gold(I)-Based Compounds as a Promising Class of Bioactive Molecules.

Breast cancers (BCs) may present dramatic diagnoses, both for ineffective therapies and for the limited outcomes in terms of lifespan. For these types of tumors, the search for new drugs is a primary necessity. It is widely recognized that gold compounds are highly active and extremely potent as anticancer agents against many cancer cell lines. The presence of the metal plays an essential role in the activation of the cytotoxicity of these coordination compounds, whose activity, if restricted to the ligands alone, would be non-existent. On the other hand, gold exhibits a complex biochemistry, substantially variable depending on the chemical environments around the central metal. In this review, the scientific findings of the last 6-7 years on two classes of gold(I) compounds, containing phosphane or carbene ligands, are reviewed. In addition to this class of Au(I) compounds, the recent developments in the application of Auranofin in regards to BCs are reported. Auranofin is a triethylphosphine-thiosugar compound that, being a drug approved by the FDA-therefore extensively studied-is an interesting lead gold compound and a good comparison to understand the activities of structurally related Au(I) compounds.

Microfluidic Formulation of DNA-Loaded Multicomponent Lipid Nanoparticles for Gene Delivery.

In recent years, lipid nanoparticles (LNPs) have gained considerable attention in numerous research fields ranging from gene therapy to cancer immunotherapy and DNA vaccination. While some RNA-encapsulating LNP formulations passed clinical trials, DNA-loaded LNPs have been only marginally explored so far. To fulfil this gap, herein we investigated the effect of several factors influencing the microfluidic formulation and transfection behavior of DNA-loaded LNPs such as PEGylation, total flow rate (TFR), concentration and particle density at the cell surface. We show that PEGylation and post-synthesis sample concentration facilitated formulation of homogeneous and small size LNPs with high transfection efficiency and minor, if any, cytotoxicity on human Embryonic Kidney293 (HEK-293), spontaneously immortalized human keratinocytes (HaCaT), immortalized keratinocytes (N/TERT) generated from the transduction of human primary keratinocytes, and epidermoid cervical cancer (CaSki) cell lines. On the other side, increasing TFR had a detrimental effect both on the physicochemical properties and transfection properties of LNPs. Lastly, the effect of particle concentration at the cell surface on the transfection efficiency (TE) and cell viability was largely dependent on the cell line, suggesting that its case-by-case optimization would be necessary. Overall, we demonstrate that fine tuning formulation and microfluidic parameters is a vital step for the generation of highly efficient DNA-loaded LNPs.

Combination of vasculature targeting, hypofractionated radiotherapy, and immune checkpoint inhibitor elicits potent antitumor immune response and blocks tumor progression.

BACKGROUND: Tumor endothelial marker 1 (TEM1) is a protein expressed in the tumor-associated endothelium and/or stroma of various types of cancer. We previously demonstrated that immunization with a plasmid-DNA vaccine targeting TEM1 reduced tumor progression in three murine cancer models. Radiation therapy (RT) is an established cancer modality used in more than 50% of patients with solid tumors. RT can induce tumor-associated vasculature injury, triggering immunogenic cell death and inhibition of the irradiated tumor and distant non-irradiated tumor growth (abscopal effect). Combination treatment of RT with TEM1 immunotherapy may complement and augment established immune checkpoint blockade. METHODS: Mice bearing bilateral subcutaneous CT26 colorectal or TC1 lung tumors were treated with a novel heterologous TEM1-based vaccine, in combination with RT, and anti-programmed death-ligand 1 (PD-L1) antibody or combinations of these therapies, tumor growth of irradiated and abscopal tumors was subsequently assessed. Analysis of tumor blood perfusion was evaluated by CD31 staining and Doppler ultrasound imaging. Immunophenotyping of peripheral and tumor-infiltrating immune cells as well as functional analysis was analyzed by flow cytometry, ELISpot assay and adoptive cell transfer (ACT) experiments. RESULTS: We demonstrate that addition of RT to heterologous TEM1 vaccination reduces progression of CT26 and TC1 irradiated and abscopal distant tumors as compared with either single treatment. Mechanistically, RT increased major histocompatibility complex class I molecule (MHCI) expression on endothelial cells and improved immune recognition of the endothelium by anti-TEM1 T cells with subsequent severe vascular damage as measured by reduced microvascular density and tumor blood perfusion. Heterologous TEM1 vaccine and RT combination therapy boosted tumor-associated antigen (TAA) cross-priming (ie, anti-gp70) and augmented programmed cell death protein 1 (PD-1)/PD-L1 signaling within CT26 tumor. Blocking the PD-1/PD-L1 axis in combination with dual therapy further increased the antitumor effect and gp70-specific immune responses. ACT experiments show that anti-gp70 T cells are required for the antitumor effects of the combination therapy. CONCLUSION: Our findings describe novel cooperative mechanisms between heterologous TEM1 vaccination and RT, highlighting the pivotal role that TAA cross-priming plays for an effective antitumor strategy. Furthermore, we provide rationale for using heterologous TEM1 vaccination and RT as an add-on to immune checkpoint blockade as triple combination therapy into early-phase clinical trials.

Evaluation of anticancer role of a novel ruthenium(II)-based compound compared with NAMI-A and cisplatin in impairing mitochondrial functionality and promoting oxidative stress in triple negative breast cancer models.

Platinum-based compounds are the most widely used anticancer drugs but, their elevated toxicity and chemoresistance has stimulated the study of others, such as ruthenium-based compounds. NAMI-A and UNICAM-1 were tested in vitro, comparing the mechanisms of toxicity, in terms of mitochondrial functionality and cellular oxidative stress. UNICAM-1, showed a clear mitochondrial target and a cytotoxic dose-dependent response thanks to its ability to promote an imbalance of cellular redox status. It impaired directly mitochondrial respiratory chain, promoting mitochondrial superoxide anion production, leading to mitochondrial membrane depolarization. All these aspects, could make UNICAM-1 a valid alternative for chemotherapy treatment of breast cancer.

Acetylshikonin isolated from Lithospermum erythrorhizon roots inhibits dihydrofolate reductase and hampers autochthonous mammary carcinogenesis in Δ16HER2 transgenic mice.

Breast cancer (BC) is the most common cancer in women and, among different BC subtypes, triple negative (TN) and human epidermal growth factor receptor 2 (HER2)-positive BCs have the worst prognosis. In this study, we investigated the anticancer activity of the root ethanolic and hexane extracts from Lithospermum erythrorhizon, a traditional Chinese herbal medicine known also as tzu ts'ao or tzu-ken, against in vitro and in vivo models of TNBC and HER2-positive BC. Treatment with L. erythrorhizon root extracts resulted in a dose-dependent inhibition of BC cell viability and in a significant reduction of the growth of TNBC cells transplanted in syngeneic mice. Acetylshikonin, a naphthoquinone, was identified as the main bioactive component in extracts and was responsible for the observed antitumor activity, being able to decrease BC cell viability and to interfere with autochthonous mammary carcinogenesis in Δ16HER2 transgenic mice. Acetylshikonin anticancer effect depends on its ability to act as a potent inhibitor of dihydrofolate reductase (DHFR), to down-regulate key mediators governing cancer growth and progression, such as HER2, Src and STAT3, and to induce apoptosis by caspase-3 activation. The accumulation of acetylshikonin in blood samples as well as in brain, kidney, liver and tumor tissues was also investigated by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) highlighting that L. erythrorhizon treatment is effective in delivering the active compound into the target tissues. These results provide evidence that L. erythrorhizon extract and in particular its main component acetylshikonin are effective against aggressive BC subtypes and reveal new acetylshikonin mechanisms of action.

Inhibition of Asaia in Adult Mosquitoes Causes Male-Specific Mortality and Diverse Transcriptome Changes.

Mosquitoes can transmit many infectious diseases, such as malaria, dengue, Zika, yellow fever, and lymphatic filariasis. Current mosquito control strategies are failing to reduce the severity of outbreaks that still cause high human morbidity and mortality worldwide. Great expectations have been placed on genetic control methods. Among other methods, genetic modification of the bacteria colonizing different mosquito species and expressing anti-pathogen molecules may represent an innovative tool to combat mosquito-borne diseases. Nevertheless, this emerging approach, known as paratransgenesis, requires a detailed understanding of the mosquito microbiota and an accurate characterization of selected bacteria candidates. The acetic acid bacteria Asaia is a promising candidate for paratransgenic approaches. We have previously reported that Asaia symbionts play a beneficial role in the normal development of Anopheles mosquito larvae, but no study has yet investigated the role(s) of Asaia in adult mosquito biology. Here we report evidence on how treatment with a highly specific anti-Asaia monoclonal antibody impacts the survival and physiology of adult Anopheles stephensi mosquitoes. Our findings offer useful insight on the role of Asaia in several physiological systems of adult mosquitoes, where the influence differs between males and females.

Multi-Targeted Anticancer Activity of Imidazolate Phosphane Gold(I) Compounds by Inhibition of DHFR and TrxR in Breast Cancer Cells.

A class of phosphane gold(I) compounds, made of azoles and phosphane ligands, was evaluated for a screening on the regards of Breast Cancer cell panels (BC). The compounds possess N-Au-P or Cl-Au-P bonds around the central metal, and they differ for the presence of aprotic or protic polar groups in the azoles and/or the phosphane moieties to tune their hydrophilicity. Among the six candidates, only the compounds having the P-Au-N environment and not displaying neither the hydroxyl nor carboxyl groups in the ligands were found active. The compounds were screened by MTT tests in SKBR3, A17, and MDA-MB231 cancer cells, and two compounds (namely the 4,5-dicyano-imidazolate-1yl-gold(I)-(triphenylphosphane, 5, and 4,5-dichloro-imidazolate-1yl-gold(I)-triphenylphosphane, 6) were found very cytotoxic, with the most active with an IC50 value of 3.46 µM in MDA-MB231 cells. By performing enzymatic assays in the treated cells lysates, the residual enzymatic activity of dihydrofolate reductase (DHFR) has been measured after cell treatment for 4 or 12 h in comparison with control cells. Upon 12 h of treatment, the activity of DHFR was significantly reduced in both SKBR3 and A17 cells by compounds 5 and 6, but not in human MDA-MB231 cells; interestingly, it was found remarkably high after 4 h of treatment, revealing a time dependence for the DHFR enzymatic assays. The DHFR inhibition data have been compared to those for the thioredoxin reductase (TrxR), the most recognized molecular target for gold compounds. For this latter, similar residual activities (i.e., 37 and 49% for the match of SKBR3 cells and compound 5 or 6, respectively) were found. Binding studies on the regards of ct-DNA (calf-thymus-DNA) and of plasma transporters proteins, such as BSA (bovine serum albumin) and ATF (apo transferrin), were performed. As expected for gold compounds, the data support strong binding to proteins (Ksv values range: 1.51 ÷ 2.46 × 104 M-1) and a weaker interaction with ct-DNA's minor groove (Ksv values range: 1.55 ÷ 6.12 × 103 M-1).

Downregulation of miR-223 Expression Is an Early Event during Mammary Transformation and Confers Resistance to CDK4/6 Inhibitors in Luminal Breast Cancer.

miR-223 is an anti-inflammatory miRNA that in cancer acts either as an oncosuppressor or oncopromoter, in a context-dependent manner. In breast cancer, we demonstrated that it dampens the activation of the EGF pathway. However, little is known on the role of miR-223 during breast cancer onset and progression. miR-223 expression was decreased in breast cancer of luminal and HER2 subtypes and inversely correlated with patients' prognosis. In normal luminal mammary epithelial cells, miR-223 acted cell autonomously in the control of their growth and morphology in three-dimensional context. In the MMTV-Δ16HER2 transgenic mouse model, oncogene transformation resulted in a timely abrogation of miR-223 expression, likely due to activation of E2F1, a known repressor of miR-223 transcription. Accordingly, treatment with CDK4/6 inhibitors, which eventually results in restraining E2F1 activity, restored miR-223 expression and miR-223 ablation induced luminal breast cancer resistance to CDK4/6 inhibition, both in vitro and in vivo. Notably, miR-223 expression was lost in microdissected ductal carcinoma in situ (DCIS) from patients with luminal and HER2-positive breast cancer. Altogether, these results identify downmodulation of miR-223 as an early step in luminal breast cancer onset and suggest that it could be used to identify aggressive DCIS and predict the response to targeted therapy. SIGNIFICANCE: miR-223 may represent a predictive biomarker of response to CDK4/6 inhibitors and its loss could identify DCIS lesions that are likely to progress into invasive breast cancer.

Subcellular Targeting of the Euplotes raikovi Kinase Er-MAPK1, as Revealed by Expression in Different Cell Systems.

In the ciliate Euplotes raikovi, a 631-amino acid Er-MAPK1 protein kinase was found to localize in nucleoli of the transcriptionally active nucleus (macronucleus) and act as a key component of an autocrine, cell-growth promoting self-signaling mechanism. While its 283-amino acid N-terminal domain includes all the structural specificities of the mitogen-activated protein kinases required for a catalytic function, the 348-amino acid C-terminal domain is structurally unique with undetermined functions. By expressing the two Er-MAPK1 domains tagged with the green fluorescent protein in mammalian fibroblasts, the yeast Schizosaccharomyces pombe and the ciliate Tetrahymena thermophila, evidence was obtained that the C-terminal domain contains all the sequence information responsible for the Er-MAPK1 subcellular localization. However, in fibroblasts and S. pombe this information determined a nucleolar localization of the GFP-tagged C-terminal domain, and a ciliary localization in T. thermophila. In the light of these findings, the Er-MAPK1 localization in E. raikovi was re-examined via immunoreactions and shown to be ciliary besides that nuclear, as is the case for the mammalian intestinal cell kinase with which the Er-MAPK1 N-terminal domain shares a strong sequence identity and a catalytic function.

A novel 3'-tRNAGlu-derived fragment acts as a tumor suppressor in breast cancer by targeting nucleolin.

tRNA-derived fragments (tRFs) have been defined as a novel class of small noncoding RNAs. tRFs have been reported to be deregulated in cancer, but their biologic function remains to be fully understood. We have identified a new tRF (named tRF3E), derived from mature tRNAGlu, that is specifically expressed in healthy mammary glands but not in breast cancer (BC). Consistently, tRF3E levels significantly decrease in the blood of patients with epidermal growth factor receptor 2 (HER2)-positive BC reflecting tumor status (control > early cancer > metastatic cancer). tRF3E down-regulation was recapitulated in Δ16HER2 transgenic mice, representing a BC preclinical model. Pulldown assays, used to search for proteins capable to selectively bind tRF3E, have shown that this tRF specifically interacts with nucleolin (NCL), an RNA-binding protein overexpressed in BC and able to repress the translation of p53 mRNA. The binding properties of NCL-tRF3E complex, predicted in silico and analyzed by EMSA assays, are congruent with a competitive displacement of p53 mRNA by tRF3E, leading to an increased p53 expression and consequently to a modulation of cancer cell growth. Here, we provide evidence that tRF3E plays an important role in the pathogenesis of BC displaying tumor-suppressor functions through a NCL-mediated mechanism.-Falconi, M., Giangrossi, M., Elexpuru Zabaleta, M., Wang, J., Gambini, V., Tilio, M., Bencardino, D., Occhipinti, S., Belletti, B., Laudadio, E., Galeazzi, R., Marchini, C., Amici, A. A novel 3'-tRNAGlu-derived fragment acts as a tumor suppressor in breast cancer by targeting nucleolin.

Cancer Vaccines Co-Targeting HER2/Neu and IGF1R.

(1) Background: Human epidermal growth factor receptor 2 (HER2)/neu-driven carcinogenesis is delayed by preventive vaccines able to elicit autochthonous antibodies against HER2/neu. Since cooperation between different receptor tyrosine kinases (RTKs) can occur in human as well as in experimental tumors, we investigated the set-up of DNA and cell vaccines to elicit an antibody response co-targeting two RTKs: HER2/neu and the Insulin-like Growth Factor Receptor-1 (IGF1R). (2) Methods: Plasmid vectors carrying the murine optimized IGF1R sequence or the human IGF1R isoform were used as electroporated DNA vaccines. IGF1R plasmids were transfected in allogeneic HER2/neu-positive IL12-producing murine cancer cells to obtain adjuvanted cell vaccines co-expressing HER2/neu and IGF1R. Vaccination was administered in the preneoplastic stage to mice prone to develop HER2/neu-driven, IGF1R-dependent rhabdomyosarcoma. (3) Results: Electroporated DNA vaccines for murine IGF1R did not elicit anti-mIGF1R antibodies, even when combined with Treg-depletion and/or IL12, while DNA vaccines carrying the human IGF1R elicited antibodies recognizing only the human IGF1R isoform. Cell vaccines co-expressing HER2/neu and murine or human IGF1R succeeded in eliciting antibodies recognizing the murine IGF1R isoform. Cell vaccines co-targeting HER2/neu and murine IGF1R induced the highest level of anti-IGF1R antibodies and nearly significantly delayed the onset of spontaneous rhabdomyosarcomas. (4) Conclusions: Multi-engineered adjuvanted cancer cell vaccines can break the tolerance towards a highly tolerized RTK, such as IGF1R. Cell vaccines co-targeting HER2/neu and IGF1R elicited low levels of specific antibodies that slightly delayed onset of HER2/neu-driven, IGF1R-dependent tumors.